The mouse mammary tumor virus (MMTV) long terminal repeat (LTR) adopts a specific nucleoprotein organization when introduced into cells. This structure involves the apparent positioning of six nucleosomes over the 1300 base pair LTR. To investigate the basis of this structure, we cross-linked histones to DNA in vivo with formaldehyde, isolated mononucleosomes after micrococcal nuclease digestion, and determined the distribution of LTR fragments in purified 146 bp length DNA. We found the A, B, and F region LTR nucleosomes each occupy a multiple set of translational positions, indicating that the low resolution phasing pattern results from the frequency-biased occupancy of a subset of these frames. This distribution of frame occupancy is highly reproducible in independently established cell lines, indicating that organization of the structure is directed by features of LTR DNA and proteins that bind to the sequence. The functional importance of this chromatin structure was tested by comparing steroid receptor induced transcription of MMTV from transient templates, which have a disordered, non-phased pattern, to the response induced from stable, replicated templates, which have the highly ordered chromatin structure. 1) Transiently introduced progesterone receptor was found to be incapable of activating the replicated structure, although quite active on transient, disorganized templates. In contrast, transiently introduced glucocorticoid receptor was functional on both transient and stable templates. Studies are underway with chimeric receptors to evaluate the region(s) of the receptors that are necessary for chromatin activation. 2) Activation of the protein kinase A phosphorylation pathway stimulated steroid activation of tran- sient templates, but suppressed expression from replicated templates. These studies confirm that the organized nucleoprotein structure found on the MMTV LTR is functionally important in gene regulation.